Abstract Brain edema and associated increase in intracranial pressure are major causes of morbidity and mortality in traumatic brain injury (TBI). Cytotoxic edema (intracellular swelling, chiefly in astrocytes) represents a major component of the brain edema in the early phase following TBI. Surprisingly, little is known about its mechanisms, and its treatment remains a therapeutic challenge. Cell swelling represents a failure of the cell to regulate its own volume as a consequence of a dysfunction in various ion transporting systems (ITSs). One ITS which appears to significantly contribute to the astrocyte swelling in TBI is the Na+-K+-2Cl- cotransporter (NKCC). Our hypothesis proposes that trauma triggers the activation of various "factors" that subsequently converge to activate NKCC, ultimately resulting in astrocyte swelling/brain edema. Among these "factors", include oxidative/nitrosative stress (ONS), mitogen-activated protein kinases (MAPKs), and the transcription factor nuclear factor-kappaB (NF-jB). Aim #1 will examine the involvement of NKCC in the astrocytic swelling post in vitro trauma as well as characterize NKCC protein alterations (oxidation, nitration, phosphorylation and changes in protein expression). Aim #2 will investigate mechanisms by which various trauma-associated "factors" (ONS, MAPKs and NF-jB) contribute to alterations in NKCC. Aim #3 will examine potential neuronal- astroglial interactions in the activation of NKCC. These studies will apply conditioned media (CM) from traumatized neurons and determine their effect on NKCC activity and cell volume in traumatized astrocytes. Should such studies indicate that CM from traumatized neurons exacerbate astrocyte swelling in traumatized astrocytes, the role of ONS, MAPKs, NF-jB in the activation of NKCC will be examined (as in Aim #2). We will also examine the involvement of glutamate, as well as K+, as these agents are known to cause astrocyte swelling and are also released by neurons following trauma. Aim #4 will perform in vivo mechanistic and therapeutic investigations in TBI-associated brain edema in rats. The mechanistic component will (a) examine the immunohistochemical distribution of phosphorylated NKCC1 (active form of NKCC1) post TBI and determine whether NKCC1 is overexpressed in astrocytes;and (b) determine whether "factor" blockers diminish such overexpression of phosphorylated NKCC1. The therapeutic component will examine the effect of the NKCC inhibitor bumetanide on traumatic brain edema. We expect that knowledge garnered from these studies will yield vital mechanistic information on the cause of astrocyte swelling post trauma that should facilitate the identification of therapeutic agents aimed at bringing about a reduction in the cytotoxic brain edema occurring in the setting of traumatic brain injury. PUBLIC HEALTH RELEVANCE: Project Narrative Brain edema is a life threatening complication associated with traumatic brain injury (TBI). Such edema in the early phase of injury is predominantly cytotoxic due to swelling of astrocytes. There is no effective therapy currently available of brain edema in TBI. We propose to examine the role of various swelling causing factors activated by trauma on astrocyte swelling/brain edema, as well as their therapeutic efficacy in vivo.